Osteoclast Differentiation Research Articles

Viscoelastic Hydrogel Modulates Phenotype of Macrophage-Derived Multinucleated Cells and Macrophage Differentiation in Foreign Body Reactions.

Biomaterial-induced macrophage-derived multinucleated cells (MNCs) are often observed at or near material implantation sites, yet their subtypes and roles in tissue repair and wound healing remain unclear. This study compares material-induced MNCs to cytokine-induced MNCs using both in vitro and in vivo models. 3D-embedded Raw264.7 cells and rat bone marrow-derived monocytes (BMDMs), with or without cytokines such as IL-4 and RANKL, were characterized for their MNC morphologies and subtypes via in situ immunocytochemistry and flow cytometry. Macrophage polarization and osteoclastic differentiation were assessed through NO production, arginase activity, and tartrate-resistant acid phosphatase levels. 3D matrix-induced MNCs expressed the same phenotypic heterogeneity as the IL-4 and RANK-treated ones. 3D matrix-induced MNCs displayed the same phenotypic heterogeneity as those treated with IL-4 and RANKL. A high viscoelastic matrix (1006.48 ± 92.29 Pa) induced larger populations of proinflammatory and osteoclast-like MNCs, whereas a low viscoelastic matrix (38.61 ± 7.56 Pa) supported active differentiation and gene expression across pro-, anti-inflammatory, and osteoclast-like macrophages. Matrix viscoelasticity also influenced the effects of IL-4 and RANKL on macrophage-derived MNC polarization. In an in vivo subcutaneous implantation model, medium to high viscoelastic matrices exhibited higher populations of CD86+ and RANK+ MNCs, while low viscoelastic matrices showed higher populations of CD206+ MNCs. These findings suggest that matrix viscoelasticity modulates macrophage differentiation and MNC phenotype, with low viscoelastic matrices potentially favoring anti-inflammatory MNCs and macrophage differentiation suitable for subcutaneous implantation.

Matrix-bound nanovesicles alleviate particulate-induced periprosthetic osteolysis.

Aseptic loosening of orthopedic implants is an inflammatory disease characterized by immune cell activation, chronic inflammation, and destruction of periprosthetic bone, and is one of the leading reasons for prosthetic failure, affecting 12% of total joint arthroplasty patients. Matrix-bound nanovesicles (MBVs) are a subclass of extracellular vesicle recently shown to mitigate inflammation in preclinical models of rheumatoid arthritis and influenza-mediated "cytokine storm." The molecular mechanism of these anti-inflammatory properties is only partially understood. The objective of the present study was to investigate the effects of MBV on RANKL-induced osteoclast formation in vitro and particulate-induced osteolysis in vivo. Results showed that MBV attenuated osteoclast differentiation and activity by suppressing the NF-κB signaling pathway and downstream NFATc1, DC-STAMP, c-Src, and cathepsin K expression. In vivo, local administration of MBV attenuated ultrahigh molecular weight polyethylene particle-induced osteolysis, bone reconstruction, and periosteal inflammation. The results suggest that MBV may be a therapeutic option for preventing periprosthetic loosening.

An Integrated Microcurrent Delivery System Facilitates Human Parathyroid Hormone Delivery for Enhancing Osteoanabolic Effect.

Human parathyroid hormone (1-34) (PTH) exhibits osteoanabolic and osteocatabolic effects, with shorter plasma exposure times favoring bone formation. Subcutaneous injection (SCI) is the conventional delivery route for PTH but faces low delivery efficiency due to limited passive diffusion and the obstruction of the vascular endothelial barrier, leading to prolonged drug exposure times and reduced osteoanabolic effects. In this work, a microcurrent delivery system (MDS) based on multimicrochannel microneedle arrays (MMAs) is proposed, achieving high efficiency and safety for PTH transdermal delivery. The internal microchannels of the MMAs are fabricated using high-precision 3D printing technology, providing a concentrated and safe electric field that not only accelerates the movement of PTH but also reversibly increases vascular endothelial permeability by regulating the actin cytoskeleton and interendothelial junctions through Ca2+-dependent cAMP signaling, ultimately promoting PTH absorption and shortening exposure times. The MDS enhances the osteoanabolic effect of PTH in an osteoporosis model by inhibiting osteoclast differentiation on the bone surface compared to SCI. Moreover, histopathological analysis of the skin and organs demonstrated the good safety of PTH delivered by MDS in vivo. In addition to PTH, the MDS shows broad prospects for the high-efficiency transdermal delivery of macromolecular drugs.

Employing single-cell RNA sequencing coupled with an array of bioinformatics approaches to ascertain the shared genetic characteristics between osteoporosis and obesity.

This study examined the relationship between obesity (OB) and osteoporosis (OP), aiming to identify shared genetic markers and molecular mechanisms to facilitate the development of therapies that target both conditions simultaneously. Using weighted gene co-expression network analysis (WGCNA), we analyzed datasets from the Gene Expression Omnibus (GEO) database to identify co-expressed gene modules in OB and OP. These modules underwent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and protein-protein interaction analysis to discover Hub genes. Machine learning refined the gene selection, with further validation using additional datasets. Single-cell analysis emphasized specific cell subpopulations, and enzyme-linked immunosorbent assay (ELISA), protein blotting, and cellular staining were used to investigate key genes. WGCNA revealed critical gene modules for OB and OP, identifying the Toll-like receptor (TLR) signalling pathway as a common factor. TLR2 was the most significant gene, with a pronounced expression in macrophages. Elevated TLR2 expression correlated with increased adipose accumulation, inflammation, and osteoclast differentiation, linking it to OP development. Our study underscores the pivotal role of TLR2 in connecting OP and OB. It highlights the influence of TLR2 in macrophages, driving both diseases through a pro-inflammatory mechanism. These insights propose TLR2 as a potential dual therapeutic target for treating OP and OB.

Butyrate: a bridge between intestinal flora and rheumatoid arthritis.

In patients with rheumatoid arthritis (RA), intestinal flora imbalance and butyrate metabolism disorders precede clinical arthritis and are associated with the pathogenesis of RA. This imbalance can alter the immunology and intestinal permeability of the intestinal mucosa, leading to damage to the intestinal barrier. In this context, bacteria and their metabolites can enter the bloodstream and reach the distant target tissues of the host, resulting in local inflammation and aggravating arthritis. Additionally, arthritis is also exacerbated by bone destruction and immune tolerance due to disturbed differentiation of osteoclasts and adaptive immune cells. Of note, butyrate is a metabolite of intestinal flora, which not only locally inhibits intestinal immunity and targets zonulin and tight junction proteins to alleviate intestinal barrier-mediated arthritis but also inhibits osteoclasts and autoantibodies and balances the immune responses of T and B lymphocytes throughout the body to repress bone erosion and inflammation. Therefore, butyrate is a key intermediate linking intestinal flora to the host. As a result, restoring the butyrate-producing capacity of intestinal flora and using exogenous butyrate are potential therapeutic strategies for RA in the future.

Circulating Baseline CXCR3+Th2 and Th17 Cell Proportions Correlate With Trabecular Bone Loss After 48 Weeks of Biological Treatment in Early Rheumatoid Arthritis.

The high prevalence of osteoporosis in rheumatoid arthritis (RA) is due to inflammation that stimulates differentiation of osteoclasts, a process involving circulating monocytes and T cell-derived factors. The aim of this study was to evaluate relations between circulating monocytes, T cell subsets, and changes in bone characteristics before and after treatment with biological disease-modifying antirheumatic drugs (bDMARDs) in RA. Thirty patients with untreated early RA who met the American College of Rheumatology/EULAR 2010 criteria were included. Data were collected before and 48 weeks after treatment with methotrexate (MTX) together with one of three bDMARDs (abatacept, tocilizumab, or certolizumab pegol). Disease activity was measured using the Clinical Disease Activity Index, swollen or tender joint counts, C-reactive protein levels, and erythrocyte sedimentation rates. Proportions of monocyte and CD4+ T cell subsets in blood samples were analyzed by flow cytometry. Bone densitometry was performed using high-resolution peripheral quantitative computed tomography (HR-pQCT). HR-pQCT revealed an overall decrease in cortical (P = 0.009) and trabecular (P = 0.034) bone mineral density, although a subset of patients showed no bone loss after 48 weeks of treatment. The overall bone loss was not associated with age, body mass index, sex, intraarticular glucocorticoid injections, or baseline disease activity. Loss of trabecular bone volume fraction correlated with high proportions of circulating CXCR3+Th2 cells (r = -0.38, P = 0.04) and CXCR3+Th17 cells (r = -0.36, P = 0.05) at baseline. Similarly, no loss of trabecular bone volume fraction correlated with high proportions of regulatory T cells (r = 0.4, P = 0.03) at baseline. However, the associations were not significant when corrected for confounders and multiple testing. MTX together with bDMARDs efficiently reduce disease activity but only prevent bone loss in a subset of patients with RA after 48 weeks of treatment. The correlations of circulating baseline T helper cell and regulatory T cell populations with trabecular bone changes suggest a potential novel role for these cells in systemic bone homeostasis during early RA.

Amentoflavone maintaining extracellular matrix homeostasis and inhibiting subchondral bone loss in osteoarthritis by inhibiting ERK, JNK and NF-κB signaling pathways

Amentoflavone (AF), a plant biflavone isolated from Selaginella sinensis ethanol extract, is characterized by anti-inflammatory and anti-oxidant properties. According to previous studies, inflammation and oxidative stress are closely related to the pathophysiology of osteoarthritis (OA). However, the effects and mechanisms of AF on OA have not been elucidated.To investigate the inhibitory effects and its molecular mechanism of AF on extracellular matrix (ECM) degradation stimulated by IL-1β as well as subchondral bone loss induced by RANKL in mice chondrocytes. Quantitative PCR was used to detect the mRNA expression of genes related to inflammation, ECM, and osteoclast differentiation. Protein expression level of iNOS, COX-2, MMP13, ADAMTS5, COL2A1, SOX9, NFATc1, c-fos, JNK, ERK, P65, IκBα was measured by western blotting. The levels of TNF-α and IL-6 in the supernatants were measured by ELISA. The amount of ECM in chondrocytes was measured using toluidine blue staining. The levels of Aggrecan and Col2a1 in chondrocytes were measured using immunofluorescence. Tartrate-resistant acid phosphatase (TRAP) staining, F-actin staining and immunofluorescence were used to detect the effect of AF on osteoclast differentiation and bone resorption. The effect of AF on destabilization of the medial meniscus (DMM)-induced OA mice can be detected in hematoxylin–eosin (H&E) staining, Safranin O green staining and immunohistochemistry.AF might drastically attenuated IL-1β-stimulated inflammation and reduction of ECM formation by blocking ERK and NF-κB signaling pathways in chondrocytes. Meanwhile, AF suppressed the formation of osteoclasts and the resorption of bone function induced by RANKL. In vivo, AF played a protective role by stabilizing cartilage ECM and inhibiting subchondral bone loss in destabilization of the medial meniscus (DMM)-induced OA mice, further proving its protective effect in the development of OA. Our study show that AF alleviated OA by suppressing ERK, JNK and NF-κB signaling pathways in OA models in vitro and DMM-induced OA mice, suggesting that AF might be a potential therapeutic agent in the treatment of OA.

Orthosilicic acid inhibits human osteoclast differentiation and bone resorption.

Silicon (Si), which is present in the diet in the bioavailable form of orthosilicic acid (OSA) and is detected as a dissolution product of certain bone-substitute materials, is suggested to promote bone health, and enhance bone healing, respectively. Silicon has been shown to stimulate osteoblastic cell differentiation and function, although the effect of Si on human osteoclasts is unclear. The present study investigated the direct effects of Si on human osteoclast differentiation, gene expression, and bone resorption. Human CD14+ monocytes were isolated from buffy coats and cultured with M-CSF and RANKL in medium without or with Si (50 μg/ml; constituting 75% OSA). The effects of Si on osteoclast differentiation were evaluated by TRAP-staining and the expression levels of CtsK, CalcR, TRAP, and DC-STAMP measured by RT-qPCR. The effect of Si on the expression level of AQP9, which encodes a potential Si transporter, was also analyzed. Bone resorption was determined based on the number of resorption pits formed when the RANKL-stimulated monocytes were cultured on bone slices, and by the levels of type I collagen fragments released into the cell culture medium. Silicon significantly inhibited the number of TRAP+ multinucleated cells and the expression of osteoclast related genes but increased the late expression of AQP9. Furthermore, Si significantly inhibited the number of resorption pits and the amount of collagen fragments in the medium when cells were cultured on bone slices. Our results demonstrate that OSA inhibits RANKL-stimulated human osteoclast differentiation, gene expression of osteoclast phenotypic markers, and bone resorption.

Measuring Calcium Levels in Bone-Resorbing Osteoclasts and Bone-Forming Osteoblasts.

Bone remodeling is a crucial, dynamic process that renews bone and maintains mineral homeostasis. It consists of several steps, including osteoclastic bone resorption and osteoblastic bone formation and mineralization. Intracellular calcium signaling is essential for osteoclast and osteoblast differentiation and activity. Here, we describe the differentiation of human osteoclasts and osteoblasts in vitro and provide common methods to determine cell differentiation and activity. We then describe protocols for measuringintracellular calcium in these cells using Fura2-AM.

Integrated analysis of single-cell RNA-seq and bulk RNA-seq revealed key genes for bone metastasis and chemoresistance in prostate cancer.

Prostate cancer (PCa) is a serious malignancy. The main causes of PCa aggravation and death are unexplained resistance to chemotherapy and bone metastases. This study aimed to investigate the molecular mechanisms associated with the dynamic processes of progression, bone metastasis, and chemoresistance in PCa. Through comprehensive analysis of single-cell RNA sequencing (scRNA-seq) data, Gene Expression Omnibus (GEO) tumor progression and metastasis-related genes were identified. These genes were subjected to lasso regression modeling using the Cancer Genome Atlas (TCGA) database. Tartrate-resistant acid phosphatase (TRAP) staining and real-time quantitative PCR (RT-qPCR) were used to evaluate osteoclast differentiation. CellMiner was used to confirm the effect of LDHA on chemoresistance. Finally, the relationship between LDHA and chemoresistance was verified using doxorubicin-resistant PCa cell lines. 7928 genes were identified as genes related to tumor progression and metastasis. Of these, 7 genes were found to be associated with PCa prognosis. The scRNA-seq and TCGA data showed that the expression of LDHA was higher in tumors and associated with poor prognosis of PCa. In addition, upregulation of LDHA in PCa cells induces osteoclast differentiation. Additionally, high LDHA expression was associated with resistance to Epirubicin, Elliptinium acetate, and doxorubicin. Cellular experiments demonstrated that LDHA knockdown inhibited doxorubicin resistance in PCa cells. LDHA may play a potential contributory role in PCa initiation and development, bone metastasis, and chemoresistance. LDHA is a key target for the treatment of PCa.

Maltobionic acid protects against ovariectomy‐induced osteoporosis by suppressing bone resorption

AbstractBackgroundOsteoporosis is a debilitating disease characterized by decreased bone density. In this study, we evaluated the anti‐osteoporotic effect of maltobionic acid (MB), one of the components of honey, and calcium maltobionate (MBCa) on bone density and metabolism using a mouse model of osteoporosis. The underlying mechanisms of MB and MBCa action were also investigated.ResultsOvariectomized (OVX) mice were fed diet containing MB or MBCa for 80 days, and femoral bone mineral content and mineral density (BMD) were evaluated. As expected, OVX reduced BMD; however, the administration of MB and MBCa significantly prevented this decrease. Furthermore, MB and MBCa treatment significantly reduced the serum levels of tartrate‐resistant acid phosphatase 5b (TRACP‐5b), a bone resorption marker, and significantly increased the levels of serum calcitonin, compared to those in the OVX control group. In vitro, the relatively high‐dose levels of MB and MBCa inhibited osteoclast differentiation by decreasing the protein expression of nuclear factor of activated T‐cell cytoplasmic 1 (NFATc1), the master transcription factor of osteoclastogenesis.ConclusionOur results showed that osteoporotic mice treated with MB and MBCa had improved bone density and bone metabolism compared to the OVX controls. Moreover, it was hypothesized herein that the suppressive effect on osteoclast differentiation slightly contributed to these results concomitantly. These findings suggest that the intake of MB or MBCa may contribute to the maintenance of bone health, including the prevention of primary osteoporosis.

Therapeutic effect of bloodletting on bone deterioration induced by hypobaric hypoxia in young rats

ObjectivesHigh-altitude regions, comprising hypoxic conditions, are associated with different altitude-induced pathologies, including a reduction in bone density. Elucidating the mechanisms underlying bone degradation in such environments and developing targeted interventions and therapeutics is important. Bloodletting therapy has promising clinical applications, but its effects on the skeletal system and bone homeostasis are not well understood. The aim of this study was to investigate the effects of a hypobaric hypoxia environment on specific femoral morphological and structural properties, including bone volume, cortical thickness, and trabecular microarchitecture, in juvenile Sprague–Dawley (SD) rats, and to explore the potential modulating effects of a bloodletting intervention on these parameters. MethodsMale SD rats, 6 weeks of age, were subjected to a simulated hypobaric hypoxia environment, replicating a 5000-m altitude, for 12 weeks. For the bloodletting intervention group, rats were subjected to a weekly 500 μL tail vein blood withdrawal. Micro-CT technology, hematoxylin and eosin staining, and tartrate-resistant acid phosphatase staining were employed to comprehensively assess the femoral microstructure, tissue architecture, and cellular morphology. Additionally, immunofluorescence analysis was conducted to quantify the expression of key proteins, and transcriptome analysis was performed to identify differentially expressed genes. ResultsExposure of rats to hypobaric hypoxia led to a significant reduction in bone mineral content, trabecular bone number, and cortical bone thickness, suggesting a deterioration of bone microstructure. Additionally, the hypoxic environment upregulated the expression of RANKL and HIF-1α, while downregulating RUNX2. Notably, although bloodletting intervention did not significantly reverse these bone structural changes, transcriptome analysis revealed its regulatory influence on the expression of key genes, particularly Mmp2, Fosl2, and URS0000B2A65A, which are implicated in pathways governing the hypoxic response, osteoclast differentiation, and PI3K–Akt signaling. ConclusionThis study highlights the detrimental effect of hypobaric hypoxia on the bone microstructure of juvenile rats and underscores the therapeutic potential of bloodletting to ameliorate this condition. Additionally, our study on the regulatory mechanisms mediating bloodletting's effects on gene expression offers fresh perspectives on bone alterations. It suggests promising avenues for the development of novel preventative measures and targeted therapies to address the challenges posed by related bone disorders.

Pdk3's role in RANKL-induced osteoclast differentiation: insights from a bone marrow macrophage model.

Osteoporosis (OP) is a chronic disease characterized by decreased bone mass, loss of skeletal structural integrity and increased susceptibility to fracture. Available studies have shown that the pyruvate dehydrogenase kinase (PDK) family is associated with osteoclastogenesis and bone loss, but the specific role of Pdk3 in bone pathology has not been systematically investigated. A cell OP model was established in receptor activator for nuclear factor-κB Ligand (RANKL)-induced bone marrow macrophages (BMMs). Hereafter, the expression levels of Pdk3 and osteoclastogenesis feature genes including nuclear factor of activated T cells 1 (Nfatc1), Cathepsin K (Ctsk), osteoclast associated Ig-like receptor (Oscar) in BMMs-derived osteoclasts were examined based on real-time quantitative PCR and western blotting methods. Further, the phosphorylation of ERK, P65 and JAK/STAT and their correlation was Pdk3 was gauged. In particular, changes in the activity of these signaling pathways were observed by silencing experiments of the Pdk3 gene (using small interfering RNA). Finally, the effects of Pdk3 gene silencing on signaling pathway activity, osteoclastogenesis, and related inflammatory and apoptotic indicators were observed by transfection with PDK3-specific siRNA. Following RANKL exposure, the levels of Pdk3 and osteoclastogenesis feature genes were all elevated, and a positive correlation between Pdk3 and osteoclastogenesis feature genes was seen. Meanwhile, ERK, P65 and JAK/STAT phosphorylation was increased by RANKL, and Pdk3 was confirmed to be positively correlated with the phosphorylation of ERK, P65 and JAK/STAT. Additionally, in RANKL-exposed osteoclasts, Pdk3 knockdown diminished the phosphorylation of ERK, P65 and JAK/STAT, reduced the expressions of osteoclastogenesis feature genes. Importantly, knockdown of Pdk3 also reduced the expression of inflammatory cytokines and resulted in elevated levels of Bax and Casp3 expression, as well as downregulation of Bcl2 expression. This study reveals for the first time the role of Pdk3 in RANKL-induced osteoclastogenesis and OP. These findings provide a foundation for future studies on the role of Pdk3 in other bone diseases and provide new ideas for the development of OP therapeutics targeting Pdk3.

Disulfiram ameliorates bone loss in ovariectomized mice by suppressing osteoclastogenesis.

Disulfiram (DSF), known as an anti-alcoholism drug, has been reported to suppress osteoclast differentiation in vitro; however, it remains uncertain whether DSF is effective in preventing osteoclastogenesis in vivo. This study aimed to investigate the effect of DSF administration in osteoporotic mice and its contribution to osteoclastogenesis in vivo. The bone phenotype of ovariectomized mice, both treated and untreated with DSF, was examined using microcomputed tomography analysis. Osteoclastic and osteoblastic parameters were assessed through bone morphometric analysis. The direct effect of DSF on osteoblastogenesis in vitro was evaluated via a primary osteoblast culture experiment. The expression of genes related to DSF targets (Nup85, Ccr2, and Ccr5) in osteoclast-lineage cells was examined using scRNA-seq analysis and flow cytometry analysis using the bone marrow cells from ovariectomized mice. The impact of DSF on osteoclast-lineage cells was assessed using primary cultures of osteoclasts. DSF administration ameliorated ovariectomy-induced bone loss and mitigated the increase of osteoclasts without affecting osteoblastogenesis. The scRNA-seq data revealed that osteoclast precursor cells expressed Nup85, Ccr2, and Ccr5. CCR2 and CCR5-positive cells in osteoclast precursor cells within bone marrow increased following ovariectomy, and this increase was canceled by DSF administration. Finally, we found that DSF had a significant inhibitory effect on osteoclastogenesis in the early stage by suppressing Tnfrsf11a expression. This study demonstrates that DSF could be a candidate for osteoporosis therapies because it suppresses osteoclastogenesis from an early stage in vivo.

Epithelial RANKL Limits Experimental Periodontitis via Langerhans Cells.

Due to its capacity to drive osteoclast differentiation, the receptor activator of nuclear factor kappa-β ligand (RANKL) is believed to exert a pathological influence in periodontitis. However, RANKL was initially identified as an activator of dendritic cells (DCs), expressed by T cells, and exhibits diverse effects on the immune system. Hence, it is probable that RANKL, acting as a bridge between the bone and immune systems, plays a more intricate role in periodontitis. Using ligature-induced periodontitis (LIP), rapid alveolar bone loss was detected that was later halted even though the ligature was still present. This late phase of LIP was also linked with immunosuppressive conditions in the gingiva. Further investigation revealed that the ligature prompted an immediate migration of RANK-expressing Langerhans cells (LCs) and EpCAM+ DCs, the antigen-presenting cells (APCs) of the gingival epithelium, to the lymph nodes, followed by an expansion of T regulatory (Treg) cells in the gingiva. Subsequently, the ligatured gingiva was repopulated by monocyte-derived RANK-expressing EpCAM+ DCs, while gingival epithelial cells upregulated RANKL expression. Blocking RANKL signaling with monoclonal antibodies significantly reduced the frequencies of Treg cells in the gingiva and prevented gingival immunosuppression. In addition, RANKL signaling facilitated the differentiation of LCs from bone marrow precursors. To further investigate the role of RANKL, we used K14-RANKL mice, in which RANKL is overexpressed by gingival epithelial cells. The elevated RANKL expression shifted the steady-state frequencies of LCs and EpCAM+ DCs within the epithelium, favoring LCs over EpCAM+ DCs. Following ligature placement, heightened levels of Treg cells were observed in the gingiva of K14-RANKL mice, and alveolar bone loss was significantly reduced. These findings suggest that RANKL-RANK interactions between gingival epithelial cells and APCs are crucial for suppressing gingival inflammation, highlighting a protective immunological role for RANKL in periodontitis that was overlooked due to its osteoclastogenic activity.

Breast Cancer Bone Metastasis Therapy and Tumor‐Associated Bone Destruction Repair by Versatile Semiconducting Nanointegrators with X‐Ray Adjuvant

AbstractTumor progress and tumor‐associated osteolysis are two key issues of breast cancer bone metastasis, which makes it challenging for bone metastasis treatment. To settle these two issues concurrently, a versatile semiconducting nanointegrator (termed as SPNCpG/Ca) containing semiconducting polymer nanoparticle (SPN), Ca2+ and cytosine‐phosphate‐guanine (CpG) oligonucleotides conjugated on the surface via a singlet oxygen (1O2)‐responsive linker, is designed. The antitumor effect can be triggered with X‐ray irradiation as an adjuvant, in which SPN works as a radiosensitizer to produce 1O2 for radiotherapy and controlled release of CpG via disrupting 1O2‐responsive linkers. The Ca2+ accumulation via SPNCpG/Ca delivering causes tumor cell death and the released CpG activates immune response to realize immunotherapy. The combinational action of radiotherapy, Ca2+ overloading, and immunotherapy results in complete clearance of metastatic tumor cells in 4T1 breast cancer‐based bone metastasis mouse models. Furthermore, Ca2+ can accelerate osteogenesis of bone marrow mesenchymal stem cells while CpG inhibits osteoclast differentiation in the bone metastasis microenvironment to alleviate osteolysis, which synergistically contributes to repair of tumor‐associated bone destruction. SPNCpG/Ca represents a versatile therapeutic nanosystem with the abilities to treat bone metastasis and repair tumor‐associated bone destruction, providing a new tactic for bone metastasis therapy.

Machine Learning Accelerates Screening of Osteoclast Differentiation Inhibitors from Natural Products.

Natural products that inhibit osteoclast differentiation are promising therapeutic and preventive agents for osteoporosis. Conventionally, identifying osteoclast differentiation involves visual inspection of the microscope images of stained osteoclasts. In this study, a supervised machine learning model was developed to classify bright-field microscope images of osteoclasts without staining. The model was used to screen a compound library, and osteoclast differentiation inhibitors were identified, demonstrating the validity of our method. Next, an in-house library of fungal extracts was screened, and pinolidoxin was revealed as an inhibitor of osteoclast differentiation. Our machine learning method enabled accurate, objective, and high-throughput evaluation of osteoclast differentiation and efficient screening of the inhibitors from natural product extracts. This study represents the first machine learning classification developed to evaluate the inhibitory activity of natural products in osteoclast differentiation.

Sporidiobolus pararoseus polysaccharides relieve rheumatoid arthritis by regulating arachidonic acid metabolism and bone remodeling signaling pathway

Rheumatoid arthritis (RA) is an autoimmune-mediated disease with the highest disability rate. Sporidiobolus pararoseus polysaccharides (SPP) have been demonstrated to have anti-rheumatoid and microbiota-modulatory effects; however, the underlying mechanisms remain unclear. This study employed collagen-induced arthritis (CIA) mice to explore the metabolic and genetic pathways. The results revealed SPP intervention significantly reduced the serum levels of rheumatoid and pro-inflammatory complement factors. SPP promoted the transition of macrophages of CIA mice toward the M2 phenotype (F4/80+/CD206+) from an inflammatory phenotype (F4/80+/CD86+) using flow cytometry analysis. A total of 44 metabolites were upregulated, and 110 metabolites were significantly downregulated by SPP compared to those in RA group. The decreased metabolites, 12(S)-HPETE, prostaglandin H2, 15-HETE, hepoxilin B3, and 15-keto-prostaglandin F2a, were mostly enriched in arachidonic acid metabolism (enrichment = 11.4 %), which was highly correlated with the anti-rheumatic activity of SPP. Gene expression analysis revealed that SPP significantly regulated OPG/RANKL/TRAF6 signaling pathway, stimulating osteogenic remodeling. Furthermore, arachidonic acid metabolism was identified as the critical metabolic driver of RA phenotypes and osteoclast differentiation, potentially associated with SPP-reshaped intestinal microbiota (i.e., Rikenellaceae_RC9_gut_group, Bacteroides, and Parabacteroides). Collectively, this study utilized an integrated approach of metabolomics and gene expression analysis to investigate the regulatory role of SPP in RA progression.

Irg1 regulates bone homeostasis via regulating the Grk5 expression

We have previously demonstrated that itaconic acid can regulate osteoclast differentiation in vitro and in vivo, thereby affecting the progression of osteoporosis. The role of Irg1 as itaconic acid catalytic enzyme in bone homeostasis has not been clearly elucidated. Here, we detected enhanced the osteoclast differentiation in Irg1-deficiency BMMs, along with the expression of genes associated with osteoclastogenesis. Irg1 knockout promoted the expression of Nfatc1 and F-actin ring formation, with the inhibited production of itaconate. RNA-seq analysis was carried out and we proved that Grk5 expression was increased the most. Inhibition of Grk5 attenuated the effect of Irg1 in the osteoclastogenesis. However, micro-CT analysis showed no significant difference of bone trabecular structure in Irg1 knockout mice. Moreover, we observed no significant difference of osteoclasts numbers in the femur of Irg1 knockout mice in vivo. And similar bone formation was detected between the Irg1 knockout and WT mice, indicating that irg1 had slight effect on the bone homeostasis under physiological conditions. Surprising, we detected higher level of inflammatory factors in the bone tissues of Irg1 knockout mice. Above all, we for the first time demonstrated that Irg1 knockout promoted the osteoclastogenesis via regulating the Grk5 signaling. Regulation of irg1-Grk5 axis could be effective in treating human diseases under pathological situations in the future.

Administration of low intensity vibration and a RANKL inhibitor, alone or in combination, reduces bone loss after spinal cord injury-induced immobilization in rats

We previously reported an ability of low-intensity vibration (LIV) to improve selected biomarkers of bone turnover and gene expression and reduce osteoclastogenesis but lacking of evident bone accrual. In this study, we demonstrate that a prolonged course of LIV that initiated at 2 weeks post-injury and continued for 8 weeks can protect against bone loss after SCI in rats. LIV stimulates bone formation and improves osteoblast differentiation potential of bone marrow stromal stem cells while inhibiting osteoclast differentiation potential of marrow hematopoietic progenitors to reduce bone resorption. We further demonstrate that the combination of LIV and RANKL antibody reduces SCI-related bone loss more than each intervention alone. Our findings that LIV is efficacious in maintaining sublesional bone mass suggests that such physical-based intervention approach would be a noninvasive, simple, inexpensive and practical intervention to treat bone loss after SCI. Because the combined administration of LIV and RANKL inhibition better preserved sublesional bone after SCI than either intervention alone, this work provides the impetus for the development of future clinical protocols based on the potential greater therapeutic efficacy of combining non-pharmacological (e.g., LIV) and pharmacological (e.g., RANKL inhibitor or other agents) approaches to treat osteoporosis after SCI or other conditions associated with severe immobilization.